Sung-Kyun Moon

Cisplatin Cytotoxicity of Auditory Cells Requires Secretions of Proinflammatory Cytokines via Activation of ERK and NF-κB

The ototoxicity of cisplatin, a widely used chemotherapeutic agent, involves a number of mechanisms, including perturbation of redox status, increase in lipid peroxidation, and formation of DNA adducts. In this study, we demonstrate that cisplatin increased the early immediate release and de novo synthesis of proinflammatory cytokines, including TNF-α, IL-1β, and IL-6, through the activation of ERK and NF-κB in HEI-OC1 cells, which are conditionally immortalized cochlear cells that express hair cell markers. Both neutralization of proinflammatory cytokines and pharmacologic inhibition of ERK significantly attenuated the death of HEI-OC1 auditory cells caused by cisplatin and proinflammatory cytokines. We also observed a significant increase in the protein and mRNA levels of proinflammatory cytokines in both serum and cochleae of cisplatin-injected rats, which was suppressed by intraperitoneal injection of etanercept, an inhibitor of TNF-α. Immunohistochemical studies revealed that TNF-α expression was mainly located in the spiral ligament, spiral limbus, and the organ of Corti in the cochleae of cisplatin-injected rats. NF-κB protein expression, which overlapped with terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling-positive signal, was very strong in specific regions of the cochleae, including the organ of Corti, spiral ligament, and stria vascularis. These results indicate that proinflammatory cytokines, especially TNF-α, play a central role in the pathophysiology of sensory hair cell damage caused by cisplatin.

Antimicrobial activity of innate immune molecules against Streptococcus pneumoniae, Moraxella catarrhalis and nontypeable Haemophilus influenzae

BackgroundDespite its direct connection to the nasopharynx which harbors otitis media pathogens as part of its normal flora, the middle ear cavity is kept free of these bacteria by as yet unknown mechanisms. Respiratory mucosal epithelia, including those of the middle ear and eustachian tube, secrete antimicrobial effectors including lysozyme, lactoferrin and β defensins-1 and -2. To elucidate the role of these innate immune molecules in the normal defense and maintenance of sterility of respiratory mucosa such as that of the middle ear, we assessed their effect on the respiratory pathogens nontypeable Haemophilus influenzae (NTHi) 12, Moraxella catarrhalis 035E, and Streptococcus pneumoniae 3, and 6B.MethodsTwo assay methods, the radial assay and the liquid broth assay, were employed for testing the antimicrobial activity of the molecules. This was done in order to minimize the possibility that the observed effects were artifacts of any single assay system employed. Also, transmission electron microscopy (TEM) was employed to evaluate the effect of antimicrobial innate immune molecules on OM pathogens. For the statistical analysis of the data, Students t-test was performed.ResultsResults of the radial diffusion assay showed that β defensin-2 was active against all four OM pathogens tested, while treatment with β defensin-1 appeared to only affect M. catarrhalis. The radial assay results also showed that lysozyme was quite effective against S. pneumoniae 3 and 6B and was partially bacteriostatic/bactericidal against M. catarrhalis. Lysozyme however, appeared not to affect the growth of NTHi. Thus, lysozyme seems to have a more pronounced impact on the growth of the Gram-positive S. pneumoniae as compared to that of Gram-negative pathogens. Lactoferrin on the other hand, enhanced the growth of the bacteria tested. The results of the radial assays were confirmed using liquid broth assays for antimicrobial activity, and showed that lysozyme and β defensin-2 could act synergistically against S. pneumoniae 6B. Moreover, in the liquid broth assay, β defensin-1 showed a modest inhibitory effect on the growth of S. pneumoniae 6B. As assessed by ultrastructural analysis, lysozyme and β defensin-2, and to a much lesser extent, β defensin-1, appeared to be able to cause damage to the bacterial membranes.ConclusionsHere we report that lysozyme and the β defensins can inhibit the growth of clinical isolates of otitis media pathogens – namely NTHi strain 12, S. pneumoniae strains 3 and 6B and M. catarrhalis strain 035E – and cause ultrastructural damage to these pathogens. Moreover, we demonstrate that lysozyme and β defensin-2 can act synergistically against S. pneumoniae. These findings are consistent with the concept that secreted antimicrobial peptides and other components of innate immunity constitute the first line of defense protecting host mucosal surfaces, including the tubotympanal (eustachian tube and middle ear cavity) mucosa, against pathogens.

Cell biology of tubotympanum in relation to pathogenesis of otitis media : a review

The sterility of the eustachian tube and tympanic cavity of normal individuals is maintained not only by the adaptive immune system, but also by the mucociliary system and the antimicrobial molecules of innate immunity. Mucin production and periciliary fluid homeostasis are essential for normal mucociliary function and dysfunction of this system is an important risk factor for otitis media. The secreted antimicrobial molecules of the tubotympanum include lysozyme, lactoferrin, beta defensins, and the surfactant proteins A and D (SP-A, SP-D). Defects in the expression or regulation of these molecules may also be the major risk factor for otitis media.

Activation of a Src-dependent Raf–MEK1/2–ERK signaling pathway is required for IL-1α-induced upregulation of β-defensin 2 in human middle ear epithelial cells

Abstract β-defensin 2 is produced by a variety of epithelial cell types in the body and exhibits potent antimicrobial activity against a variety of pathogens, including the bacteria that are most commonly associated with otitis media (OM). The human β-defensin 2 (hBD-2) gene is an NF-κB regulated gene and a variety of proinflammatory stimuli can induce its expression. Although the presence of molecules of innate immunity such as lysozyme and lactoferrin has been demonstrated in the middle ear, to date there have been no reports on the expression of β-defensin 2. In the present study, we demonstrate that β-defensin 2 is expressed in the middle ear mucosa of humans and rats. We also show that it is expressed in a human middle ear epithelial cell line and that its expression is induced by proinflammatory stimuli such as interleukin 1 alpha (IL-1α), tumor necrosis factor alpha (TNF-α), and lipopolysaccharide (LPS). Moreover, we demonstrate that the transcriptional activation of hBD-2 gene by IL-1α is mediated through an Src-dependent Raf–MEK1/2–ERK signaling pathway.

Evidence that Cisplatin-induced Auditory Damage is Attenuated by Downregulation of Pro-inflammatory Cytokines Via Nrf2/HO-1

Recently, we demonstrated that pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 played a critical role in cisplatin-induced cochlear injury and that flunarizine, known as a T-type Ca2+ channel antagonist, induced a cytoprotective effect against cisplatin cytotoxicity in HEI-OC1 cells by the activation of NF-E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) cascade through PI3K-Akt signaling but calcium-independent pathway. We report here that flunarizine markedly attenuates cisplatin-induced pro-inflammatory cytokine secretion and their messenger RNA transcription as well as cisplatin cytotoxicity through the activation of Nrf2/HO-1 and downregulation of NF-κB. In HEI-OC1 cells, overexpression of Nrf2/HO-1 by gene transfer or pharmacological approaches attenuated cisplatin-induced cytotoxicity and pro-inflammatory cytokine production. On the contrary, inhibition of Nrf2/HO-1 signaling by pharmacological inhibitors or specific small interfering RNAs significantly abolished the beneficial effects of flunarizine. Flunarizine also attenuated cisplatin-mediated MAPK activation and pharmacological inhibition of MAPKs, especially MEK1/ERK, blocked cisplatin-induced NF-κB activation in HEI-OC1 cells. Furthermore, WT-Nrf2 overexpression effectively blocked MAPK activation after cisplatin exposure. Finally, orally administrated Sibelium™, the trade name of flunarizine, suppressed the increase of pro-inflammatory cytokines by cisplatin in both serum and cochleas of mice, whereas it increased HO-1 expression in cochleas. These results indicate that flunarizine induces a protective effect against cisplatin ototoxicity through the downregulation of NF-κB by Nrf2/HO-1 activation and the resulting inhibition of pro-inflammatory cytokine production in vitro and in vivo.

Immortalization of Normal Adult Human Middle Ear Epithelial Cells Using a Retrovirus Containing the E6/E7 Genes of Human Papillomavirus Type 16:

A human middle ear epithelial cell line (HMEEC-1) was established using human papillomavirus E6/E7 genes. HMEEC-1 has remained morphologically and phenotypically stable, even after 50 passages. The cells are anchorage-dependent and nontumorigenic when injected into nude mice. This cell line thus provides a new tool for the study of normal cell biology and the pathological processes associated with the epithelial cells of the middle ear in otitis media. HMEEC-1 will also be useful in the search for new drugs and biological agents for the treatment of otitis media.

Spiral ligament fibrocytes release chemokines in response to otitis media pathogens

Conclusion. Spiral ligament fibrocytes (SLFs) may be involved in the innate immune response of the inner ear by producing chemoattractants for recruiting inflammatory cells such as neutrophils and monocytes. Objective. The purpose of this study was to investigate the cellular responses of SLFs when challenged by inflammatory stimuli such as components of otitis media pathogens or proinflammatory cytokines. Materials and methods. To detect released inflammatory cytokines and chemokines, cells were treated for 48 h with whole lysates of nontypable Haemophilus influenzae (NTHi), Streptococcus pneumoniae, or with interleukin 1 alpha (IL-1α). The culture medium was then collected and applied to protein arrays. To compare mRNA levels of chemokines, total RNA was extracted after 3 h of treatment with the above agents, and quantitative real-time PCR was performed. Results. Protein array analysis showed that in response to NTHi or S. pneumoniae, rat SLFs released monocyte chemotactic protein 1, macrophage inflammatory protein 3 alpha, TNF-α, and cytokine-induced neutrophil chemoattractant 2 and 3. Treatment with IL-1α, on the other hand, resulted in release of MCP-1 but not the other molecules. Tissue inhibitor of metalloproteinase 1 and vascular endothelial growth factor were released regardless of the inflammatory stimulus used.

Synergistic effect of interleukin 1 alpha on nontypeable Haemophilus influenzae-induced up-regulation of human beta-defensin 2 in middle ear epithelial cells.

BackgroundWe recently showed that beta-defensins have antimicrobial activity against nontypeable Haemophilus influenzae (NTHi) and that interleukin 1 alpha (IL-1 alpha) up-regulates the transcription of beta-defensin 2 (DEFB4 according to new nomenclature of the Human Genome Organization) in human middle ear epithelial cells via a Src-dependent Raf-MEK1/2-ERK signaling pathway. Based on these observations, we investigated if human middle ear epithelial cells could release IL-1 alpha upon exposure to a lysate of NTHi and if this cytokine could have a synergistic effect on beta-defensin 2 up-regulation by the bacterial components.MethodsThe studies described herein were carried out using epithelial cell lines as well as a murine model of acute otitis media (OM). Human cytokine macroarray analysis was performed to detect the released cytokines in response to NTHi exposure. Real time quantitative PCR was done to compare the induction of IL-1 alpha or beta-defensin 2 mRNAs and to identify the signaling pathways involved. Direct activation of the beta-defensin 2 promoter was monitored using a beta-defensin 2 promoter-Luciferase construct. An IL-1 alpha blocking antibody was used to demonstrate the direct involvement of this cytokine on DEFB4 induction.ResultsMiddle ear epithelial cells released IL-1 alpha when stimulated by NTHi components and this cytokine acted in an autocrine/paracrine synergistic manner with NTHi to up-regulate beta-defensin 2. This synergistic effect of IL-1 alpha on NTHi-induced beta-defensin 2 up-regulation appeared to be mediated by the p38 MAP kinase pathway.ConclusionWe demonstrate that IL-1 alpha is secreted by middle ear epithelial cells upon exposure to NTHi components and that it can synergistically act with certain of these molecules to up-regulate beta-defensin 2 via the p38 MAP kinase pathway.

Effects of Retinoic Acid, Triiodothyronine and Hydrocortisone on Mucin and Lysozyme Expression in Cultured Human Middle Ear Epithelial Cells

Mucous hypersecretion is a major complication of otitis media and can prolong the disease course and increase morbidity. Mucin, a major component of mucus, is a macromolecular complex of glycoprotein and makes mucus viscous. Lysozyme is a secretory element of the middle ear mucosa, which has a non-specific and innate antibacterial function. We attempted to identify factors that regulate these secretory products and their morphological phenotype using cultured human middle ear epithelial cells. Cellular differentiation was induced by creating an air-liquid interface on culture day 9 in serum-free conditioned media. Omission of retinoic acid (RA) caused decrease in the secretion of mucin and lysozyme, and in the cellular expression of MUC 2, MUC 5AC and MUC 5B mRNA. In contrast, removal of triiodothyronine (T3) caused an increase in the secretion of mucin and the level of MUC5AC mRNA. When hydrocortisone (HC) was removed from the media, the secretion of mucin was decreased without an apparent change of message level. The expression of MUC 1 mRNA was not changed by the respective deficiency of RA, T3 or HC. The effect of T3 or HC on lysozyme was not significant. This study shows that RA, T3 and HC influence the morphological phenotype and the secretory function of mucin and lysozyme in cultured human middle ear epithelial cells. This culture system can serve as an in vitro model for study of the regulation of various cellular secretions in human middle ear epithelium.Mucous hypersecretion is a major complication of otitis media and can prolong the disease course and increase morbidity. Mucin, a major component of mucus, is a macromolecular complex of glycoprotein and makes mucus viscous. Lysozyme is a secretory element of the middle ear mucosa. which has a non-specific and innate antibacterial function. We attempted to identify factors that regulate these secretory products and their morphological phenotype using cultured human middle ear epithelial cells. Cellular differentiation was induced by creating an air liquid interface on culture day 9 in serum-free conditioned media. Omission of retinoic acid (RA) caused decrease in the secretion of mucin and lysozyme, and in the cellular expression of MUC 2, MUC 5AC and MUC 5B mRNA. In contrast, removal of triiodothyronine (T3) caused an increase in the secretion of mucin and the level of MUC5AC mRNA. When hydrocortisone (HC) was removed from the media, the secretion of mucin was decreased with out an apparent change of message level. The expression of MUC 1 mRNA was not changed by the respective deficiency of RA. T3 or HC. The effect of T3 or HC on lysozyme was not significant. This study shows that RA, T3 and HC influence the morphological phenotype and the secretory function of mucin and lysozyme in cultured human middle ear epithelial cells. This culture system can serve as an in vitro model for study of the regulation of various cellular secretions in human middle ear epithelium.

Panel 4: Recent Advances in Otitis Media in Molecular Biology, Biochemistry, Genetics, and Animal Models

Background Otitis media (OM) is the most common childhood bacterial infection and also the leading cause of conductive hearing loss in children. Currently, there is an urgent need for developing novel therapeutic agents for treating OM based on full understanding of molecular pathogenesis in the areas of molecular biology, biochemistry, genetics, and animal model studies in OM. Objective To provide a state-of-the-art review concerning recent advances in OM in the areas of molecular biology, biochemistry, genetics, and animal model studies and to discuss the future directions of OM studies in these areas. Data Sources and Review Methods A structured search of the current literature (since June 2007). The authors searched PubMed for published literature in the areas of molecular biology, biochemistry, genetics, and animal model studies in OM. Results Over the past 4 years, significant progress has been made in the areas of molecular biology, biochemistry, genetics, and animal model studies in OM. These studies brought new insights into our understanding of the molecular and biochemical mechanisms underlying the molecular pathogenesis of OM and helped identify novel therapeutic targets for OM. Conclusions and Implications for Practice Our understanding of the molecular pathogenesis of OM has been significantly advanced, particularly in the areas of inflammation, innate immunity, mucus overproduction, mucosal hyperplasia, middle ear and inner ear interaction, genetics, genome sequencing, and animal model studies. Although these studies are still in their experimental stages, they help identify new potential therapeutic targets. Future preclinical and clinical studies will help to translate these exciting experimental research findings into clinical applications.